Method of fixing nitrogen utilizing azotobacter oleovorans



United States Patent 3,393,063 METHOD OF FIXING NITROGEN UTILIZING AZOTOBACTER OLEOVORANS Vernon F. Coty, Trenton, N..I., and John B. Davis, Dallas, Tex., assignors to Mobil Oil Corporation, a corporation of New York No Drawing. Filed Sept. 30, 1964, Ser. No. 400,568 Claims. (Cl. 71--7) ABSTRACT OF THE DISCLOSURE A method of fixing nitrogen is disclosed. A microbe, Azotobacter oleovorans fixes nitrogen without the need of carbohydrates using hydrocarbons as an energy source.

This invention relates to nitrogen fixation. More particularly, it relates to the conversion of nitrogen into fertilizers by the action of certain microbes on hydrocarbons in the presence of elemental nitrogen.

The fixation of atmospheric or gaseous nitrogen by certain microorganisms is known. For example, aerobic bacteria of the genus Azotobacter, certain anaerobic bacteria, particularly the Clostridium pasteurianum, the Rhizobia in the root nodules of the legumes and certain blue-green algae have been demonstrated to convert nitrogen into microbial protein nitrogen. While recent work has shown the cultivation of hydrocarbon oxidizing microbes in the presence of gaseous, elemental nitrogen but in the absence of other nitrogen compounds, it is desired to extend the applicability of such a process to other hydrocarbons and to other microorganisms.

Accordingly, it is an objective of this invention to provide a process in which certain hydrocarbons can be oxidized with accompanying fixation of nitrogen. Another aim is the provision of a microorganism capable of growing on such hydrocarbons and on elemental nitrogen as the only extraneous nitrogen source. These and other purposes appear in the following description and examples, not limitative, but given for illustrative purposes only.

The goals of this invention are accomplished by the finding of new species of microorganisms which has been named Azotobacter oleovomns, n. sp. This organism grows well in the presence of hydrocarbons, for example, n-tetradecane, with gaseous, elemental nitrogen as its only source of nitrogen for conversion to organic nitrogen compounds within its cells or extra-cellularly. The fixation of elemental nitrogen occurs smoothly as the microorganism, contained in an oxidator, is fed oxygen, the hydrocarbon and nitrogen admixed with oxygen or fed separately. A healthy cellular growth and division occurs and nitrogen compounds are produced. These appear mainly as part of the microbial protein or other organic materials. A bacterial slime is also produced. Thus, both intraand extra-cellular products are produced. These may be used separately or combined as fertilizers.

From the above it can readily be appreciated that the processes involved are chemical processes or manners of new manufacture requiring an operator who maintains appropriate conditions and drives the microorganisms to the desired results. The operator separates the resultant mixtures or the desired products for further utilization as desired.

The Northern Utilization Research and Development Division of the US. Department of Agriculture, Peoria, Illinois has designated the organisms as follows: Azotobacter oleovorans, NRRL B-3145.

The invention will be further understood by reference to the examples and description below. In the examples, parts and percentages given are by weight unless otherwise noted.

Example I A number of nitrogen deficient mineral agar plates were seeded with an oil-contaminated soil and supplied with n-tetradecane as a carbon source for microbial growth. After 2 months incubation with elemental nitrogen as the only source of nitrogen, growths were found.

Transfers were effected using standard purification techniques and following this an inoculation was made into 50 ml. of nitrogen-free mineral medium containing ntetradecane. After 3 days of agitation in the presence of 0.5 cc. of n-tetradecane a growth of about 10' cells per ml. was noted. According to their morphology those belonged to the genus of Azotobacter. They were named Azototlacter oleovorans, n. sp.

Some of the cells were examined under the microscope for characterization. They were found to be coccoid in shape and motile by means of flagella. The cells were gram-negative, and, of course, aerobic. While they varied somewhat in size and pigmentation, they were all substantially about 0.5 to 1.2 by 1.7 to 11 microns in size. They were quite similar to Azotobacter i/zdicus, and the cells had at each end a highly refractive body round in shape. However, they were not slimy in appearance. While Azotobacter indicus fixes nitrogen, it does so only when using a carbohydrate such as sucrose or glucose. The Azotobacter oleov rans of this invention, however, not only fixes nitrogen without the need of carbohydrates but uses a hydrocarbon as an energy source.

Example II Following a 3 days growth in a 50 ml. system of Example I, the 50 ml. was transferred to a New Brunswick fermenter containing 3 liters of an aqueous nutrient standard in all respects save that it was nitrogen-free. It contained 3.825 g. of n-tetradecane as a substrate and two additional aliquots of the same amount were added, each at a different time, as the experiment progressed. After two weeks of incubation under agitation, 4.3 g. of cells was obtained, and apparently all of the hydrocarbon was consumed. The isolated cells contained 25% lipids and 11% protein, the remainder being carbohydrates.

The protein value represents an increase of fixed nitrogen of 25 micrograms per ml. This constituted further proof of nitrogen fixation since standard proof for nitrogen fixation requires only 5 to 10 micrograms per ml. in synthetic media.

The triglyceride fraction was found to contain C C C and C acids and, surprisingly, the odd numbered acids, C13, C15 and C17.

As to the presence of polymer, it was found that the lipid fraction contained 3% of polymeric materials. This percentage decreased as the chain length of the hydrocarbon substrate increased. Thus, for polymer production short chain aliphatics are fed.

To demonstrate the applicability of this invention to other hydrocarbons, the experiment in Example II may be repeated using n-butylbenzene. An oxidator is charged with 2 liters of an aqueous substrate containing the following salts, in the amounts given in parentheses being gram/liter: Na HPO (0.3),.KH PO (0.2), MgSO .7H O (0.1), FeSO .7H O (0.005) and Na MoO .2H O (0.002). These salts are typical of those used in standard aqueous nutrients used in microbial growth, the entire aqueous substrate being, of course, free of nitrogen. To the sub strate is added a small inoculum of the microbes from one of the transfer plates prepared in Example I. The composite is then stirred while air containing n-butylbenzene vapors is fed into the stirred mixture. The gas stream coming from the oxidator may be discarded, though in other instances it is recycled or passed to another oxidator or to traps for recovery of the hydrocarbon. The composite is kept at room temperature, being about 25 C.30 C., and the flow rate of the gaseous stream is about 0.5 liter per minute. Turbidity appears quite rapidly and growth is good. In the absence of the elemental nitrogen little or no growth results.

In another run using n-dodecane similar results are obtained.

If it is desired to demonstrate the use of a mixture of hydrocarbons, the experiment in Example II may be repeated using a mixture of n-butane and hexylcyclohexane. Recycling is used with addition of the hydrocarbon to the reentry stream periodically to keep an adequate food source circulating. Very good growth with nitrogen-fixation is attained.

Usually it is desired to increase rates of production. To demonstrate that the microorganism of this invention may grow and accomplish the objects of this invention under violent agitation, the oxidator used in Example II is equipped with a sparger and an impeller, there being also present shearing blades. The gaseous mixture is expelled through the sparger, and the stirrer is used to force the mass against the shearing surfaces. Since many of the hydrocarbons useful in this invention are solids or well-bodied liquids, it is desired to prevent them from occluding the microbial cells. This is accomplished by imparting a violent agitation to the mixture of gas, liquid and solids to the point that the size of the hydrocarbon particles is reduced to 10 microns or less. It is necessary, of course, that the microbial cells be capable of living and growing under these conditions.

To test this, a mixture of hexylcyclohexane and mineral oil is used as the hydrocarbon substrate. Using the aqueous nutrient described in and along with the other conditions of Example II but violently agitating the mass (impeller speeds of about 1800 rpm.) to effect the said size reduction, it is noted that microbial growth not only occurs but is much faster, and the yield of organic nitrogen compounds is substantially increased.

The hydrocarbons that can be used in this invention are the aliphatics and aromatics, the aromatics having, preferably, at least one aliphatic side chain containing at least 4 carbon atoms. Thus, the process of the invention can be employed for the oxidation of the alkyl substituted benzenes and the alkyl substituted 5- and 6-membered cyclo paraffins. The cyclic hydrocarbons can contain a single ring but may also contain more than one ring.

One, or two or more, alkyl substituents can be on the ring portion of the cyclic hydrocarbon. It is preferred that the alkyl substituents be those containing four, or more, carbon atoms. The alkyl substituent can be a straight-chain or a branched-chain substituent.

Alkyl substituted aromatic cyclic hydrocarbons which may be oxidized by the process of the invention include the isomeric butylbenzenes, the isomeric amylbenzenes, the isomeric hexylbenzenes, the isomeric heptylbenzenes, and the isomeric octylbenzenes.

Included among the alkyl substituted naphthenes which may be treated by the processes of the invention are the isomeric butylcyclopentanes, the isomeric amy1cyc1open tanes, the isomeric hexylcyclopentanes, the isomeric heptylcyclopentanes, and the isomeric octylcyclopentanes. Also included among these naphthenes are the isomeric butylcyclohexanes, the isomeric amylcyclohexanes, the isomeric hexylcyclohexanes, the isomeric heptylcyclohexanes, and the isomeric octylcyclohexanes.

The aliphatic hydrocarbons which may be used include methane, ethane, propane, n-butane, iso-butane, the isomeric pentanes, the isomeric hexanes, the isomeric heptanes and octanes, and higher hydrocarbons as tridecane, tetradecane, hexadecane, eicosane, tetracosane, and intermediates and isomers thereof. Also, oily materials such as mineral oil and the kerosene, fuel oils and waste hydrocarbonaccous materials may be used. Unsaturated hydrocarbons such as ethylene, propylene and cyclohexene,

4,v among others, may also be used, and the aromatics may have unsaturated side chains.

The fermentation reaction mixture should also contain, in accordance with conventional practice in carrying out microbiological reactions, mineral salts for the growth of the oxidative microorganism. These salts should furnish potassium, ferrous or ferric, calcium, magnesium, phosphate and sulfate ions, as well as ions of trace elements such aszinc, manganese, copper, and molybdenum.

The fermentation reaction mixture, during the oxidative reaction, is maintained under conditions to insure optimum growth of the fermentation microorganism. The temperature, for example, should be maintained between about 20 and about 55 C. Preferably, the temperature should be maintained in the neighborhood of 30 C. Further, the pH of the reaction mixture should be maintained near neutrality, namely, at about 7.0. However, the fermentation reaction may be carried out at a pH between about 5.5 and 8.5.

The fermentation reaction mixture will consist primarily of water. The water may constitute 99%, or more, by weight of the liquid phase of the fermentation reaction mixture. However, the water may constitute a much lesser portion of the fermentation reaction mixture. For example, the fermentation reaction mixture may contain as little as 50% by weight of water. Generally, any proportion of water heretofore employed in microbial oxidation of hydrocarbons may be used.

The microbial oxidation reaction requires that oxygen be supplied to the fermentation reaction mixture. Oxygen can be supplied to the fermentation reaction mixture by employing reactions open to the atmosphere. With agitation of the reaction mixture, the surface thereof exposed to the atmosphere is continuously being renewed and oxygen is thereby taken up by the mixture. If desired, oxygen may be supplied by bubbling oxygen, or any oxygen-containing gas such as air, through the fermentation reaction mixture. Further, if desired, for the purpose of avoiding excessive evaporation of water from the fermen-tation reaction mixture, the oxygen or oxygen-containing gas may be humidified prior to bubbling through the fermentation reaction mixture. It will be realized that, where oxygen or oxygen-containing gas is bubbled through the reaction mixture, the bubbling of the gas may provide the desired agitation of the reaction mixture. On the other hand, agitation by other means may additionally be employed in order to insure that the agitation is adequate.

Following the fermentation reaction, the various oxidation products of the cyclic hydrocarbons are removed by conventional methods from the fermentation reaction mixture. The fermentation reaction mixture may be subjected to such procedures as may be required to remove the :body cells of the microorganism. Suitable procedures include decantation, centrifuging, and filtration. The mixture may also be subjected to such procedures as may be required to remove any remaining hydrocarbon. Extraction of the reaction mixture with a solvent, such as petroleum ether, in which the hydrocarbon, but not certain oxidation products, is soluble may .be employed. 'I hereafter the reaction mixture maybe extracted with a solvent in which said oxidation products are soluble. With water contained in the reaction mixture, this solvent will be a water-immiscible solvent. Satisfactory results have been obtained by extracting the fermentation reaction mixture with diethylether or other solvent employed by evaporation or other suitable procedure. Also, oxidation products may also be recovered from the reaction mixture by steam distillation. It is also possible to use the entire aqueous mass as a fertilizer or to remove the water from it and use the residue as a fertilizer.

In any of the processes it is preferred to keep the pH neutral. Thus, while the pH may be about 6.0 to about 8.0, it is preferred to keep it at 7.0. The phosphates, such as potassium dihydrogen phosphate, are used as buffers to do this. The fermenters can be run on a continuous basis, the cells and/or slime being removed while leaving some as an inooulate and while adding fresh medium.

When gases or vapors are being used as the substrates, the concentration of the hydrocarbon is not critical. Generally, it is 15% to 50% of the hydrocarbon/ air mixture. While oxygen is required for the oxidation no special means for supplying it are needed. Thus, the fermenter may simply get its oxygen and the nitrogen to be fixed merely from the surrounding air by normal exposure of the mass to the air. Usually, it is preferred to feed air along with the hydrocarbon since this leads to greater growth by increased contact of the cells with the food.

When liquid hydrocarbons are being fed, it is preferred to use lower concentrations and this is also true for the solids. The concentrations are about 0.1% to about 1.0% based upon the weight of the medium. While concentrations may be increased by using stepwise additions and vigorous agitation, the organisms grow very well in the absence of such steps, and accordingly, normal conditions are usually used.

The process of this invention is useful in the production of an organic fertilizer. The cells and other products produced can be added to soil or the hydrocarbon can be fed to soils to which have been added Azo'tobacter oleovorans, separately or admixed with other additives, including other microorganisms. In this procedure, the hydrocarbons are generally added in amounts about 0.10% to about 0.1% by weight of the soil, but the exact amount will vary depending upon soil, weather and similar conditions. Many soils are very low in nitrogen content, and a process of this invention is directed to increasing soil nitrogen. Thus, one will seek to create a :growth situation by adding a relatively large amount of a given hydrocarbon to the soil or by adding a relatively large amount of microbial inoculum or by adding both. In this connection it is to be appreciated that cellular matter added to the soil may contain dead or live cells or mixtures thereof and the soil additive may include the culture solution or what remains of it upon evaporation of the water for the media usually contain nitrogenous matter derived from autolyzed cells or possible extra cellular products in addition to salts purposely present. The fixed nitrogen in the solution usually represents about of the total amount fixed. Further, carbohydrates and lipids are also produced. These as well as the protein material, can be used as fertilizers to add nitrogen to the ground by adding them to soils containing conventional nitrogen fixers that feed on coventional substrates.

The microorganism and compositions containing cells thereof and the processes of this invention are particularly useful in petroleum m ulches. It is known that the laying down of a petroleum coating, such as asphalt, on the ground keeps moisture within the ground beneath the coating. Such coatings are placed over seed beds leaving the areas between seed rows exposed to receive normal railfall. Water moves late-rally from the unseeded area to contiguous seeded, covered areas and is held there by the coating and is available [for use by the seed or the plant therefrom for longer than normal time periods. The coating is of such a nature that it can be pierced by the germinating seed and growing plant. Such mulches are used also with means to divert runoff water from seeded contributing areas to seeded benched areas which means may include confining means other than petroleum mulches. Applying the principles of this invention to such muches comprises applying to the soil the Azotobacter oleovoran's of the invention plus the hydrocarbonaceous substrate on which they feed. While the organism of this invention is able to feed directly on a given petroleum product, such as a petroleum mulch, a petroleum resin, or asphalt, it is beneficial to add hydrocarbons to the ground or to the petroleum coating, preferably the former. By these procedures, mulches of enhanced value are attained, since they provide not only for water retention but for nitrogen fixation. Land in arid regions is generally very low in nitrogen content. Such land may be reclaimed by use of the processes of this invention.

The processes of this invention are advantageous in that odd-numbered carboxylic acids can be obtained without regard to feeding special substrates. Further, non-volatile or only slightly volatile hydrocarbons are now available for adding to soils in the fertilization by the nitrogen fixation route.

While the invention has been disclosed herein in connection with certain embodiments and certain procedural details, it is clear that changes, modifications or equivalents can be used by those skilled in the art; accordingly, such changes within the principles of this invention are intended to be included within the scope of the claims below.

We claim:

1. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Azo tobacter oleovorwns; cultivating said cells to produce a culture thereof; forming an aqueous mixture that is a nutrient for microorganisms and that is substantially nitrogen-free; placing said aqueous mixture into a vessel; bringing elemental nitrogen, oxygen, a hydrocarbon containing aliphatic carbon atoms and some of said cells of Azotobacter oleovorans into contact with each other in said aqueous mixture contained in said vessel; and allowing said cells to metabolize said hydrocarbons and nitrogen, said Azotobacter oleovorans being numbered by the U5. Agricultural Research Laboratory as follows: NRRL B-3145.

2. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Azotobacter oleovorans; cultivating said cells to produce a culture thereof; admixing in a vessel a hydrocarbon containing aliphatic carbon atoms, elemental nitrogen, oxygen and some of said cells of Azotobacter oleovorans; and allowing said organism to grow on said hydrocarbon with said elemental nitrogen as its only source of nitrogen for growth, thereby fixing said elemental nitrogen, said Azotobacter oleovorans being numbered by the g..1A5gricultural Research Laboratory as follows: NRRL 3. A process in accordance with claim 2 in which said hydrocarbon is an alkane.

4. A process in accordance with claim 2 in which said hydrocarbon is a substituted naphthenic hydrocarbon.

5. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Azotobacter oleovorans; cultivating said cells to produce a culture thereof; forming in a vessel a mixture of a hydrocarbon containing aliphatic carbon atoms, elemental nitrogen, oxygen and some of said cells of Azotobacter oleovorans which cells oxidize said hydrocarbon and fix elemental nitrogen; keeping the resultant mixture at a pH of about 6.0 to about 8.0; and allowing said microorganism to grown on said mixture, thereby fixing said elemental nitrogen, said Azotobacter oleovorans being numbered by the U.S. Agricultural Research Laboratory as follows: NRRL B-3145.

6. A process in accordance with claim 5 which is carried out under normal atmospheric conditions of temperature and pressure.

7. A process for the fixation of nitrogen which comprises isolating from soil cells of a microorganism now known as Azotobacter oleovorans; cultivating said cells to produce a culture thereof; forming a mixture of nitrogen, oxygen and a hydrocarbon; feeding said mixture to some of said cells of Azotobacter oleovorans contained in a vessel; and allowing said cells to metabolize said hydrocarbon and nitrogen, said Azotobacter oleovorans being numbered by said US. Agricultural Research Laboratory as follows: NRRL B-3145.

8. A process in accordance with claim 7 in which the cells are in the presence of an aqueous nutrient during said feeding and said metabolization.

9. A process in accordance with claim 5 in which said nitrogen and said oxygen are fed together as air.

10. A process for increasing soil nitrogen which comprises isolating from soil cells of a microorganism now known as Azotobacter oleovorans; cultivating said cells to produce a culture thereof; adding a hydrocarbon and some of said cells to a second soil; exposing the resultant modified soil to nitrogen; and effecting fixation of elemental nitrogen by allowing said added cells to grow on said hydrocarbon, oxygen and nitrogen thereby converting said nitrogen into compounds, said Azotobucter oleorvorans being numbered by the US. Agricultural Research Laboratory as follows: NRRL B-3145.

References Cited UNITED STATES PATENTS 3,210,179 10/1965 Davis et al. 71-7 0 S. LEON BASHORE, Primary Examiner.

DONALL H. SYLVESTER, Examiner.

R. BAJEFSKY, Assistant Examiner.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No 3 ,393 ,063 July 16 1968 Vernon F. Coty et a1.

It is certified that error appears in the above identified patent and that said Letters Patent are hereby corrected as shown below:

Column 5, line 29, "0.10%" should read 0.01% line 49,

"coventional" should read conventional line 58, "railfall" should read rainfall line 68, "muches" should read mulches Column 6, line 59, "grown" should read grow Signed and sealed this 30th day of December 19690 (SEAL) Attest: Edward M. Fletcher, Jr. WILLIAM E. SCHUYLERTYIR;

Attesting Officer Commissioner of Patents 

